1α,25(OH)2VD3 is the most active form of VD3 in animals and it plays an important role in regulating mineral metabolism but also reproduction and immunity. Testes are the main reproductive organs of male mammals. In order to study the effect of 1α,25(OH)2VD3 on early testicular development, 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 μg.kg-1 of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides, 60 days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labeling technique was used to analyze the protein profile of the testes in each group. Western blotting was used to verify the target of differentially abundant proteins (DAPs). The results showed that of the identified 132715 peptides, 122755 were specific peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs was focused on proteins closely related to the regulation of the testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results also indicate that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets’ testes. Significance: The early development of the testes of breeding boars is closely related to their lifetime fertility. Our research identifies potential signaling pathways and differentially abundant proteins associated with testicular development. These findings indicate that plants containing 1α,25(OH)2VD3-glycosides used as a nutritional ingredient may help early testicular development in breeding boars and reveal the regulatory mechanism of 1α,25(OH)2VD3 on testicular development.